1. Field
This invention is generally directed to identifying protein-ligand interactions, and specifically with peptide ligands which bind human Factor VIII and which may be used in a process for the affinity purification of human Factor VIII.
2. Background
Human Factor VIII, (antihemophilic factor; FVIII:C) is a human plasma protein consisting of 2 polypeptides (light chain molecular weight of 80,000 daltons and heavy chain molecular weight variable from 90,000 to 220,000). It is an essential cofactor in the coagulation pathway; required for the conversion of Factor X into its active form (Factor Xa). Factor VIII circulates in plasma as a non-covalent complex with von Willibrand Factor (aka FVIII:RP). Blood concentrations of Factor VIII below 20% of normal cause a bleeding disorder designated hemophilia A. Factor VIII blood levels less than 1% result in a severe bleeding disorder, with spontaneous joint bleeding being the most common symptom. Factor VIII can be isolated from either a plasma derived source (cryoprecipitate) or from a genetically engineered recombinant source. Recombinant DNA technology has allowed construction of plasmids that direct the expression of fusion products of Factor VIII protein in transfected mammalian cells (See U.S. Pat. No. 4,757,006).
Several methods have been described for purification of Factor VIII from plasma sources. Tuddenham et al. disclose a method for separating FVIII from human plasma by immunoabsorbent chromatography (Tuddenham, et al., J. Laboratory Clin. Med., (1979), 93: 40-53). The method involved using rabbit anti-FVIII:C polyclonal antibodies adsorbed to agarose beads, and desorption using a calcium gradient. Notably, it was sufficiently selective to distinguish FVIII:C from FVIII:RP. D.E.G. Austen demonstrated another technique using ion-exchange chromatography on amino-hexyl-substituted agarose beads (Austin, D. E. G., British J. Heamotol., (1979), 43: 669-674). However, it has been reported that both methods suffer from some level of contamination of the resulting FVIII:C by FVIII:RP.
Zimmerman, et al., U.S. Pat. No. RE32011 disclose a human monoclonal antibody-based immunoaffinity two-step purification procedure. The first step is adsorption of the FVIII:C/FVIII:RP complex from a human plasma source, followed by a buffer wash, and subsequent desorption of FVIII:C using a calcium solution that elutes only FVIII:C (FVIII:RP remains bound). The second step is concentration of the FVIII:C by adsorbing the eluate from step one to an aminohexyl-substituted agarose column with a subsequent calcium buffer wash. This results in a concentration of FVIII of over 160,000-fold from plasma.
Several purification schemes utilize antibody affinity columns. Wood, W., et al., Nature, (1984), 312: 330-337 demonstrated another immunoaffinity purification, using the C7F7 MAb which is specific to the FVIII:C 80 kD fragment. Rotblat, F., et al., Biochemistry, (1985), 24: 4294-4300 also describe an immunoaffinity purification of FVIII:C of over 300,000-fold from cryoprecipitate by polyelectrolyte purification, followed by affinity separation of a sepharose-anti-FVIIIR:Ag, and a final adsorption to a FVIII:C specific MAb column. To date, the most successful purifications of Factor VIII from plasma or from recombinant sources have been accomplished by using murine monoclonal antibodies specific to either Factor VIII or von Willibrand Factor (see Zimmerman, et al, supra, U.S. Pat. No. RE32011).
Although monoclonal antibodies have been used successfully to obtain a relatively pure Factor VIII preparation, monoclonal antibodies can be present in the Factor VIII effluent because of leaching from the support matrix. This raises the possibility of antigenicity when the final preparation is introduced into animal systems, since murine monoclonal antibodies have been shown to be antigenic. A second disadvantage of the use of monoclonal antibodies is the requirement of cell culture facilities for producing the antibodies and the concomitant cost of purification and attachment onto a support matrix. And finally, the stability of the antibody binding site may not be amenable to the rigorous conditions necessary to sanitize the column.
Affinity chromatography is one of the most efficient techniques for purifying a protein from a complex mixture. With potential advantages including high stability, efficiency, selectivity, and low price, peptides have been investigated as affinity ligands in the pharmaceutical industry. A recent approach for identifying such ligands is to screen peptides from combinatorial peptide libraries (Baumbach, et al., BioPharm, (1992), 5: 23-35; Buettner, J., et al., Int. J. Pept. Prot. Res., (1996), 47: 70-83; Huang, P., et al., Biotechnol. & Bioeng., (1995), 47: 288-297; Huang, P., et al., Bioorg. & Med. Chem., (1996), 4: 699-708). It has been shown that by using the "divide-couple-recombine" approach (Furka, et al., Int. J. Pept. Prot. Res., (1992), 37: 487-493; Lam, et al., Nature, (1991), 354: 82-84; Houghten, et al., Nature, (1991), 354: 84-86), millions of unique peptides of a defined length may be synthesized on resin beads. Each bead contains a unique peptide sequence. These library beads and their corresponding peptide sequences are then exposed to a target protein. Among these millions of peptide sequences, the target protein may bind to several unique bead-sequences. Those beads and their corresponding sequences must be detected, isolated, and identified. Several detection systems, including colorimetric two-step methods (Buettner, et al., (1996), 47: 70-83; Houghten, et al. (1991); Lam, et al., J. Immunol. Meth., (1995), 180: 219-223) as well as direct fluorescence detection methods (Meldal, et al., Proc. Nat. Acad. Sci., (1994), 91: 3314-3318; Meldal, et al., J. Chem. Soc. Perkin Trans., (1995) 1: 1591-1596; Needles, et al., Proc. Natl. Acad. Sci., (1993), 90: 10700-10704) have been used.
Another method of generating large libraries of peptides for affinity separations is phage display. WO 95/18830, Inhibitors of human plasmin derived from the kunitz domains, describes the selection of binding domains specific for human plasmin useful as inhibitors, including applications for drugs, for diagnostic reagents and for affinity purification ligands. The claimed binding domains are chimeras of one of the three kunitz binding domains found in lipoprotein-associated coagulation inhibitor (LACI), a 39 kd protein. The binding domains are about 58 amino acids with 7 to 11 amino acid substitutions generated by combinatorial methods. No smaller peptides are used or claimed.
WO 97/35197, Purification of tissue plasminogen activator (tPA), claims binding domains useful for purification of the biological drug tPA, using the same technology as described in WO 95/18830 and WO 97/35196. In this application, the binding domains are 29 amino acids of which 8 were combinatiorialized. One claimed sequence is from a peptide of 11 amino acids, of which 8 residues were derived combinatorially from the binding motifs of the other claimed domains, having the 11-mer sequence: Arg Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa. Specific residues are claimed for each position (Xaa) for this presumed cyclic peptide.
It is apparent that other, more specific affinity peptides for binding proteins of biological interest are needed.